Of the 215,000 new cases of breast cancer that will be diagnosed in the United States in 2006, about 25% will over-express the HER2/neu proto-oncogene. Only about half of these patients will benefit from administration of the humanized anti-HER2/neu monoclonal antibody (mAb)trastuzumab with chemotherapy and none will be cured. There is no clear mechanism of action for trastuzumab therapy although it is possible that innate immune cells bearing receptors (R) for the Fc (or "constant") region of immunoglobulin are involved. We hypothesized that co-stimulation of these FcR-bearing cells with specific activating factors would significantly enhance the immune response to Ab-coated tumor cells. Indeed, we found that treatment of natural killer (NK) cells with interleukin-21 (IL-21) and immobilized IgG led to synergistic production of interferon-gamma (IFN-K) and T cell-attracting chemokines as compared to cells treated with IL-21 or IgG alone. Co-stimulation of NK cells in this manner via the IL-21R and FcpRllla led to prolonged activation of the mitogen-activated protein (MAP) kinase Erk (which was critical for NK cell cytokine secretion) and synergistic induction of the AP-1 transcription factor. We also found that the synergistic production of IFN-K by co-stimulated NK cells was dependent upon the presence of specialized signaling platforms within the NK cell membrane called lipid rafts. Our murine studies showed that IL-21 significantly enhanced the anti-tumor activity of a murine anti-HER2/neu breast cancer mAb and that this effect was dependent upon endogenously-produced IFN-K. Other cytokines have been employed in combination with mAbs with modest success, but the unique actions of IL-21 make it a superior choice for use in the setting of trastuzumab therapy: IL-21 is well-tolerated, is able to activate both NK cells and CDS* T cells, and induces a unique array of immune activating cytokines. In this proposal we will investigate the role of lipid rafts and Erk-induced AP-1 in stimulating IFN- y gene expression and the mechanism by which IFN-y promotes survival in mice receiving IL-21 and the anti-HER2 mAb. We also plan to conduct a clinical trial of IL-21 in combination with trastuzumab/paclitaxel in patients with metastatic and locally-advanced breast cancers. Patient tumors and peripheral blood immune cells will be evaluated before and after treatment in order to identify the mechanisms responsible for tumor eradication. The pre-clinical and clinical experiments proposed in this project are designed to elucidate the specific mechanisms by which administration of IL-21 enhances the activity of trastuzumab. This information should lead to further clinical trials that will test the efficacy of this therapeutic combination in patients with HER2 (+) breast cancer. We believe that these studies will identify new strategies for modulating NK cell activation in response to Ab- coated targets and that this information will lead to improvements in the mAb therapy of cancer.